Differential induction of phosphatidylcholine hydrolysis, diacylglycerol formation and protein kinase C activation by epidermal growth factor and transforming growth factor-α in normal human skin fibroblasts and keratinocytes

Abstract

We have investigated coupling between the epidermal growth factor (EGF) receptor and the phospholipase C (PLC)/protein kinase C (PKC) signal-transduction system in normal skin fibroblasts and keratinocytes, for which EGF and transforming growth factor alpha (TGF-alpha) are mitogenic. EGF and TGF-alpha induced a rapid increase in tyrosine phosphorylation of the EGF receptor, in both fibroblasts and keratinocytes, but failed to induce tyrosine phosphorylation of PLC-gamma 1 or detectable phosphoinositide hydrolysis, as measured by two sensitive assays. In fibroblasts, EGF induced phosphatidylcholine (PC) hydrolysis, resulting in increased diacylglycerol (DAG). In contrast, in keratinocytes, there was no detectable PC hydrolysis or elevation of DAG in response to EGF or TGF-alpha. EGF and TGF-alpha activated PKC in fibroblasts, as evidenced by increased phosphorylation of a specific cellular PKC substrate (myristoylated alanine-rich C-kinase substrate, 'MARCKS'). In keratinocytes, TGF-alpha and EGF induced only a modest increase in MARCKS protein phosphorylation. This apparent modest activation of PKC, in the absence of detectable DAG formation, may have been mediated by arachidonic acid, which was released from keratinocytes in response to TGF-alpha, and has been shown to stimulate PKC activity in vitro. These data demonstrate that (1) in dermal fibroblasts and keratinocytes, which express normal levels of EGF receptors, EGF receptor activation is not coupled to tyrosine phosphorylation of PLC-gamma 1 or PtdIns hydrolysis, suggesting that these events are not required for the mitogenic activity of EGF or TGF-alpha in these cells, (2) coupling of EGF receptor to PC hydrolysis is cell-type specific, and (3) in skin fibroblasts, DAG, formed through EGF-induced PC hydrolysis, is capable of activating PKC.

The human papillomavirus type 16 E5 protein modulates phospholipase C-γ-1 activity and phosphatidyl inositol turnover in mouse fibroblasts

“…Since our results show that in our cells PLC-g-1 is insensitive to EGF treatment, other mechanisms must be responsible for the permanent activation found in E5 cells. In this context it should be mentioned that in several cell systems, src-kinase can also be involved in the activation of PLC-g-1 activity (Khare et al, 1997;Reynolds et al, 1993;Singer et al, 1997). At this stage, however, whether this enhanced activation is the result of increased phosphorylation in an E5-dependent manner or an impaired PLC-g-1-speci®c phosphatase remains to be demonstrated.…”

c-Jun–dependent inhibition of cutaneous procollagen transcription following ultraviolet irradiation is reversed by all-trans retinoic acid

“…Primary cultured adult human skin fibroblasts (25) were transfected with 2 µg COL1A2 reporter-gene plasmid (-772 to +58) (26) alone or together with 100 ng expression vectors for either wild-type or dominant-negative mutant c-Jun (TAM-67) (27) using FuGene6 (Boehringer Mannheim, Indianapolis, Indiana, USA). All cells were cotransfected with pXJ40-LacZ plasmid (2 µg), containing the β-galactosidase gene.…”

Key role of PLC-γ in EGF protection of epithelial barrier against iNOS upregulation and F-actin nitration and disassembly

“…That PKC is downstream from PLC-␥ in the cell's protective cascade is also indicated by our previous finding that PKC activators (OAG or TPA) can maintain both cytoskeletal integrity and intestinal barrier function even in the presence of PLC inhibition. Indeed, PKC has also been shown to be downstream of PLC in other systems as well (47,49,51,61). Also, Fig.…”

“…PLC-␥ hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP 2 ) to produce not only inositol 1,4,5-trisphosphate (IP 3 ) but also diacyl glycerol (DAG) in epithelial cells (6,8,23,49,51,57). DAG is one of the bestcharacterized products of PLC-␥-mediated reactions and that is known to lead to downstream activation of serine/thereonine protein kinase C (PKC) (8,11,12,28,47).…”